JP2002541797A - New recombinant herpesviruses and mutant herpesviruses - Google Patents

Abstract

(57) SUMMARY The present invention provides methods and reagents for inducing active immunity in animals. In particular, the present invention provides a recombinant herpesvirus having foreign DNA capable of inducing immunity to herpesvirus, and / or a source of the foreign DNA. The invention also provides mutant herpesviruses having portions of the genome that have been deleted. Preferably, in the UL54.5 open reading frame of avian herpesvirus or the UL43 open reading frame of Marek's disease virus, exogenous DNA is introduced or a part of the genome is deleted.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to viral vectors for vaccination of animals. Specifically, the present invention relates to a viral vector having a gene insertion site for introducing foreign DNA.

BACKGROUND OF THE INVENTION Marek's disease is a chicken lymphoproliferative disease caused by Marek's disease virus (MDV). MDV (a naturally occurring herpes virus) can infect chicken sac-derived and thymus-derived lymphocytes and subsequently induce lymphomas of the thymus-derived lymphocytes. MDV is one name of the avian herpesvirus family. For example, MDV1 is a pathogenic strain of herpes virus in chickens, MDV2 is a naturally attenuated herpes virus strain in chickens, and MDV3 is a non-pathogenic herpes virus of turkey.

Since Marek's disease is a contagious disease, the virus has become an important chicken pathogen, especially in large breeding environments (eg, poultry farming). Currently, Marek's disease
Embryos at day 17-19 of the eggs or one-day-old chickens (one-day-old c
hick) vaccination.

[0004] In general, the application of recombinant DNA technology to animal viruses has a recent history. The first virus to be manipulated is the virus with the smallest genome. For example, in the case of papovaviruses, their use in genetic engineering has been as defective replicons since these viruses are very small and cannot accommodate much extra DNA. Therefore, expression of foreign DNA sequences from these viruses requires a wild-type helper virus and is restricted to cell culture systems. On the other hand, for adenovirus, there is a small amount of non-essential DNA that can be replaced by foreign sequences. This technique has also been applied to portions of the herpesvirus genome in avian herpesviruses (Cochr.
See US Patent No. 5,853,733 to An et al.).

[0005] The example of gene deletion or insertion into herpes virus demonstrates that it is possible to genetically manipulate the herpes virus genome by recombinant DNA technology. In the past, methods that have been used to insert genes include homologous recombination between viral DNA cloned into a plasmid and purified viral DNA transfected into the same animal cell. . However, the extent to which the location of the deletion and the site for insertion of the foreign DNA sequence can be generalized is not known from these previous studies.

[0006] The identification of the appropriate DNA sequence insertion site in the avian herpesvirus serves for the development of new vaccines. However, (i) the appropriate virus and (ii)
The selection of a particular portion of the genome to use as an insertion site for creating a vector for exogenous DNA sequence expression poses a significant challenge. In particular, the insertion site must be non-essential for viable replication of the virus and its operation in tissue culture and in vivo. In addition, the insertion site must be able to accept the new genetic material while at the same time ensuring that the virus continues to replicate.

[0007] What is needed is the identification of a gene insertion site for the production of new viruses and new viral vectors.

SUMMARY OF THE INVENTION [0008] The present invention provides mutants and recombinant herpesviruses comprising a foreign DNA sequence inserted into a site within the herpesvirus genome. In one embodiment,
The site is non-essential for virus replication. In a preferred embodiment,
Foreign DNA sequences can be expressed in host cells infected with the recombinant herpesvirus and its expression. In a particularly preferred embodiment, the foreign DNA sequence is also under the control of a promoter located upstream of the foreign DNA sequence. The invention is not limited to a specific site for insertion or deletion. In one embodiment, the deletion and / or insertion is in the UL54.5 open reading frame of Marek's disease virus. In another embodiment, the deletion and / or insertion is in the UL43 open reading frame of Marek's disease virus. In a preferred embodiment, the insert is in the genome of Marek's disease virus type 1.

[0009] Although not limited to a particular type of inserted DNA, in one embodiment of the invention, the foreign DNA sequence inserted into the herpesvirus genome encodes a polypeptide. Preferably, the polypeptide is immunogenic to the animal into which the recombinant herpesvirus is introduced. Preferably, the immunogenic polypeptide is a linear polymer in which more than 10 amino acids are linked by peptide bonds that stimulate the animal to produce antibodies. In a preferred embodiment, the foreign DNA sequence also encodes a detectable marker. Preferably, the detection marker is E. coli. coli B-galactosidase.

[0010] In a preferred embodiment, the recombinant herpes virus is chicken anemia virus (CAV), infectious Farbikius disease virus (IBDV), Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious laryngeal tracheitis. Includes foreign DNA sequences encoding immunogenic polypeptides derived from the virus (ILTV) or infectious bronchitis virus (IBV), fragments thereof and / or substantially homologous sequences. In another preferred embodiment, the foreign DNA encodes a cytokine. The invention also contemplates a recombinant herpes virus having more than one foreign DNA sequence encoding one or more antigens.

[0011] Where the foreign DNA sequence of the recombinant herpesvirus of the invention encodes an immunogenic polypeptide derived from infectious Farbikius disease virus (IBDV), the immunogenic polypeptide may be IBVD, VP2, VP3 or VP4. Preferably, the protein, its fragments and / or substantially homologous sequences. Outpatient D
If the NA sequence encodes an MDV-derived immunogenic polypeptide, preferably the immunogenic polypeptide is MDV glycoprotein B (gB), glycoprotein D (gD), or glycoprotein A (gA) , Fragments thereof and / or substantially homologous sequences.

If the foreign DNA sequence encodes an immunogenic polypeptide from Newcastle disease virus (NDV), the immunogenic polypeptide may be an NDV fusion (F) protein or NDV hemagglutinin neuraminidase (HN), Preferably, it is a fragment and / or a substantially homologous sequence.

If the foreign DNA sequence encodes an immunogenic polypeptide from infectious laryngeal tracheitis virus (ILTV), the immunogenic polypeptide may be ILTV glycoprotein “B” (gB), ILTV glycoprotein D (GD), or ILTV glycoprotein I (gI), a fragment thereof and / or a substantially homologous sequence.

If the foreign DNA sequence encodes an infectious bronchitis virus (IBV) derived immunogenic polypeptide, the immunogenic polypeptide may be an IBV spike protein, an IBV matrix protein, a nucleocapsid protein, a fragment thereof and / or Alternatively, it is preferably a substantially homologous sequence.

[0015] The expression of the inserted foreign DNA sequence can be controlled by a promoter located upstream of the foreign DNA sequence. Preferably, the promoter is a herpes virus promoter. More preferably, the promoter is a pseudorabies virus (PRV) gX promoter, MDV gB promoter, MDV gA promoter, MDV gD promoter, ILTV gB promoter, ILTV
gD promoter, ILTV gI promoter, human cytomegalovirus virus (HCMV) immediate early promoter and / or substantially homologous sequences.

The present invention further provides homologous vectors for producing a recombinant herpesvirus by inserting a foreign DNA sequence into the herpesvirus genome. 1
In one embodiment, the homologous vector comprises a double-stranded DNA molecule consisting essentially of a double-stranded foreign DNA sequence, at one end of the foreign DNA sequence and on one side of a non-essential site of the herpesvirus genome. A double-stranded DNA having a double-stranded DNA homologous to the located genomic DNA and having, at another end of the foreign DNA sequence, homology to a herpesvirus genomic DNA sequence located on the other side of the same site It has DNA. In such embodiments, the double-stranded DNA is contained within the BamHI "B" genomic fragment of Marek's disease virus type 1, 321
It may be homologous to a DNA sequence present within a two base pair SacI to BglII subfragment. Preferably, the DNA sequence corresponding to the promoter is
It is located upstream of the NA sequence and controls its expression. Similarly, the foreign DNA sequence preferably encodes an immunogenic polypeptide (eg, a polypeptide described above).

In one embodiment of the invention, the double-stranded herpesvirus DNA comprises MDV
Homologous to the DNA sequence present in the BamHI "B" fragment of the herpesvirus genome. Preferably, the double-stranded herpesvirus DNA is homologous to a DNA sequence present in the open reading frame encoding the UL43 protein of the herpesvirus genome. In another embodiment of the invention, the double-stranded herpesvirus DNA is homologous to a DNA sequence present in a BamHI "M" fragment of the herpesvirus genome. Preferably, the double-stranded herpesvirus DNA is homologous to a DNA sequence present in the UL54.5 gene coding region of the herpesvirus genome.

The invention further provides a vaccine comprising an immunizing-effective amount of a recombinant or mutant herpesvirus of the invention and a suitable carrier.

The present invention further provides a method of immunizing an animal. The preferred animal to be immunized is a chicken.

The invention also provides a method of immunizing a chicken in vivo. For the purposes of the present invention, this involves immunizing chickens against infectious Farbicius disease virus, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus or infectious bronchitis virus. Preferably, the method comprises the step of administering to the chicken a dose of the vaccine of the invention that effectively immunizes. The vaccine can be prepared by methods well known to those skilled in the art (eg, intramuscular injection, subcutaneous injection,
Either by intraperitoneal injection or intravenous injection). Alternatively, the vaccine may be administered intranasally, orally or intraocularly.

The present invention also provides a host cell infected with the recombinant herpesvirus of the present invention. Preferably, the host cell is a bird cell.

Definitions For the purposes of the present invention, a "host cell" is a cell used to amplify a vector and its insert. Infection of the cells can be performed by methods known to those of skill in the art (eg,
The following DNA Transformation For Generating R
ecombinant Herpesvirus 11).

The term “animal” refers to an organism in the animal kingdom. Thus, the term
Includes humans as well as other organisms. Preferably, the term refers to a vertebrate. More preferably, the term refers to animals that are birds.

An “immunizing effective amount” of a recombinant herpesvirus of the invention is in the range of 10 ² to 10 ⁹ plaque forming units (PFU) / dose.

For the purposes of the present invention, a “homology vector” is a plasmid constructed to insert a foreign DNA sequence at a specific site on the genome of a herpes virus.

An “exogenous DNA sequence” is a segment of DNA that has been joined or joined to another DNA molecule using recombinant technology, wherein this particular DNA segment naturally Not found in connection with DNA molecules. The source of such exogenous DNA may or may not be from an organism from which the DNA is located. The foreign DNA can also be a synthetic sequence with codons different from the native gene. Examples of recombinant technology include D
Examples include, but are not limited to, the use of restriction enzymes and ligases to join NAs.

An “insertion site” is a restriction site in a DNA molecule into which foreign DNA can be inserted.

A “replicable virus” is, as found in this wild-type organism,
A virus in which the genetic material contains all of the DNA or RNA sequences required for virus replication. Thus, a replication-competent virus does not require a second virus or cell line to replace what is missing or missing from the virus in order to replicate. "Non-essential site in the herpesvirus genome" means a region in the herpesvirus genome where the polypeptide product of that region is not required for viral infection or replication.

A “vector” is a replicon, such as a plasmid, phage, cosmid or virus, into which another DNA sequence may be ligated to achieve expression of the ligated DNA sequence.

“Double-stranded DNA molecule” refers to a polymeric form of a deoxyribonucleotide (adenine, guanine, thymine, or cytosine) in its normal double-stranded helix. The term refers only to the primary and secondary structure of the molecule, and is not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found in linear DNA molecules (eg, restriction fragments of DNA from viruses, plasmids and chromosomes).

A DNA “coding sequence” is a DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (eg, mammalian) DNA, viral DNA, and even synthetic DNA sequences. Not done. The polyadenylation signal and transcription termination sequence are 3
'Can be placed in.

A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase or auxiliary protein in a cell and initiating transcription of a downstream (3 ′ direction) coding sequence. For purposes of the present definition, a promoter sequence is one in which the translation initiation codon (ATG) of the coding sequence is proximate the 3 'end and that facilitates transcription at a detectable level above background. Are spread upstream (5 ′ direction) to include the minimum number of bases or elements required for Within the promoter sequence are found a transcription initiation site and a protein binding domain (consensus sequence) responsible for binding RNA polymerase. Eukaryotic promoters often (but not always) include "TATA" boxes and "CAAT" boxes, which are conserved sequences found in many eukaryotic promoter regions.

[0033] RNA polymerase interacts directly or indirectly with the promoter sequence and causes transcription of the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. If the code array is
Either "operably linked" to a control sequence or "under control" of a control sequence in a cell.

[0034] Two polypeptide sequences are "substantially identical" when at least about 80% (preferably, at least about 90%, and most preferably, at least about 95%) of the amino acids match over a defined length of the molecule. Are substantially homologous. "

[0035] The two DNA sequences are either identical or have 40 nucleotides of their nucleotides.
%, More preferably more than about 20% of the nucleotides, and most preferably no more than about 10% of the nucleotides,
"Substantially homologous".

A virus in which a foreign DNA sequence has been inserted into its genome is a “recombinant virus”, whereas a virus in which a portion of its genome has been removed by deliberate deletion (eg, genetic manipulation). Is a "mutant virus."

The term “polypeptide” is used in its broadest sense (ie, any polymer of amino acids joined by peptide bonds (more than a dipeptide)). Thus, the term “polypeptide” includes proteins, oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.

“Antigenic” refers to the ability of a molecule comprising one or more epitopes to stimulate an animal or human immune system to produce a humoral and / or cellular antigen-specific response. Say. An “antigen” is an antigenic polypeptide.

An “immunological response” to a composition or vaccine is the development of a cell- and / or antibody-mediated immune response in a host against a composition or vaccine of interest. Usually, such a response occurs when antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells are specifically directed against the antigens contained in the composition or vaccine of interest by the subject. To be produced.

The terms “immunogenic polypeptide” and “immunogenic amino acid sequence” are used to neutralize viral infectivity and / or mediate antibody complement-dependent or antibody-dependent cytotoxicity. Refers to each polypeptide or amino acid sequence that elicits an antibody that confers protection on the immunized host. As used herein, an "immunogenic polypeptide" comprises the full-length (or near full-length) sequence of a desired protein or immunogenic fragment thereof.

By “immunogenic fragment” is comprised of one or more epitopes, thus
A fragment of a polypeptide that neutralizes viral infectivity and / or mediates antibody complement-dependent cytotoxicity or antibody-dependent cytotoxicity to confer protection on an immunized host. Such fragments are usually at least about 5 amino acids in length, and preferably at least about 10 to 15 amino acids in length. There is no critical upper limit on the length of the fragment, which may include approximately the entire length of the protein sequence, or may even include a fusion protein that includes fragments of two or more antigens.

By “infectious” is meant having the ability to deliver a viral genome to a cell.

The term “open reading frame” or “ORF” is defined as the gene coding region that, when expressed, can produce a complete protein and a particular polypeptide chain protein for a particular gene.

The term “avian herpesvirus” refers to a herpesvirus that can replicate in an avian host and does not replicate naturally in other host animals.

DETAILED DESCRIPTION OF THE INVENTION Throughout this disclosure, various publications, patents and patent applications have been referenced. The disclosures of these publications, patents and patent applications are incorporated herein by reference.

The methods and compositions of the present invention include the steps of modifying cloned DNA sequences from various prokaryotic and eukaryotic sources by insertions, deletions, changes in one or more bases, And subsequently inserting these altered sequences into the genome of the herpes virus. One example is cloning a portion of herpes virus DNA into a plasmid in a bacterium, reconstructing the viral DNA in a cloned state so that the viral DNA contains a deletion of a particular sequence,
And / or additionally, adding a foreign DNA sequence either at the site of the deletion or at a site other than the deletion. The methods and compositions of the present invention also include deletion of a portion of the herpesvirus genome that produces the mutant virus.

Generally, a foreign gene construct is cloned into a nucleotide sequence corresponding to only a portion of the entire herpesvirus genome. The nucleotide sequence may have one or more appropriate deletions. The chimeric DNA sequence is usually in a plasmid that allows successful cloning to produce multiple copies of the sequence. The cloned foreign gene construct can then be included in the entire viral genome, for example, by in vivo recombination following a DNA-mediated co-transfection technique. The multiple copies of the coding sequence or more than one coding sequence may be
It can be inserted so that it can express more than one foreign protein. Foreign genes can have additions, deletions or substitutions to enhance the expression and / or immunological effects of the expressed protein.

[0048] To effect successful expression of the gene, the gene can be inserted into an expression vector along with a suitable promoter containing enhancer elements and polyadenylation sequences. Methods for constructing a number of eukaryotic promoters and polyadenylation sequences and expression cassettes that provide for successful expression of foreign genes in mammalian cells are known in the art, for example, in US Pat. No. 5,151,267. It is. The promoter is selected to provide optimal expression of the immunogenic protein, which is then used to generate a humoral immune response, according to known criteria,
It satisfactorily elicits cell-mediated and mucosal immune responses.

Foreign proteins produced by in vivo expression in recombinant virus-infected cells can themselves be immunogenic. More than one foreign gene can be inserted into the viral genome, resulting in successful production of more than one effective protein.

Thus, one utility of using a mutant herpesvirus or adding a foreign DNA sequence to the herpesvirus genome is to vaccinate animals. For example, a mutant virus can be introduced into an animal to elicit an immune response against the mutant virus.

Alternatively, a recombinant herpesvirus having a foreign DNA sequence inserted into the genome encoding the polypeptide may also be a foreign herpesvirus, a foreign DNA sequence, in an animal.
It may act to elicit an immune response against the polypeptide encoded by the sequence and / or the herpes virus. Such viruses can also be used to introduce foreign DNA and its products into a host animal to reduce defective genomic status in the host animal. These recombinant herpesviruses are referred to as viral vectors if they are viruses that can carry foreign DNA in the host animal.

[0052] The present invention is not limited to the use of a particular herpes vector. One suitable avian herpesvirus for use as a viral vector is MDV. To provide MDV as a vector and vaccine against Marek's disease, the MDV is not essential for the replication and function of the virus within the MDV genome and contains therein the MDV
It may be desirable to insert one or more endogenous genes encoding the antigen to locate a site that can further stimulate an immune response to the encoded antigen. On the other hand, MD as a viral vector or expression vector for use as a multivalent vaccine
To provide V, one or more endogenous genes are inserted into the MDV genome that are not essential for viral replication and function and that encode antigens of a poultry pathogen other than MDV. Thus, it is desirable to locate sites that can further stimulate the immune response to MDV and such other poultry pathogens. Alternatively, a copy of the endogenous gene and a combination of copies of the endogenous gene can be inserted into a non-essential region of such a viral vector.

When an MDV genome is used, it is preferred to use an attenuated type 1 MDV strain. Rispens CVI-988 is an attenuated 1 serotype MDV vaccine strain that can be used to provide protection against highly toxic strains of MDV.

The MDV genome is a linear 180 kb base pair double-stranded molecule consisting of two unique regions (a unique short region (US) and a unique long region (UL)).
Each unique region consists of an inverted repeat (long terminal repeat (TRL) and internal long inverted repeat (IRL) for UL, and a short internal inverted repeat (IRS) and short terminal repeat for US). (TRS)).

The present invention is not limited to specific DNA deletion and / or DNA insertion sites, whereas the UL43 and UL54.5 regions of the avian herpesvirus are
It has been found to contain appropriate sites for deletions and insertions. For example, there is an XhoI site in the UL43 region of the avian herpesvirus, especially in the MDV genome. There are also open reading frames (ORFs) flanking the UL54 and UL55 regions. This ORF, designated UL54.5, contains an appropriate NdeI site for deletions and insertions.

In particular, there is a 3212 base pair SacI-BglII partial fragment contained within the BamHI “B” genomic fragment of Marek's disease virus type 1. B
3212 base pairs SacI-B contained within the amHI "B" genomic fragment
Preferred deletion and / or insertion sites within the partial fragment of glII are:
Located in the open reading frame encoding herpes virus UL43, and the preferred insertion in the open reading frame is Xho.
I restriction endonuclease site.

Similarly, deletions and / or insertions can be made in the BamHI “
M "genomic fragment. A preferred insertion site within the BamHI "M" genomic fragment is located in the open reading frame encoding herpesvirus UL54.5, and a preferred insertion site within the open reading frame is an NdeI restriction endonuclease site. In a particularly preferred embodiment, the product of the UL54.5 open reading frame is not essential for viral replication.

Various foreign DNA or coding sequences (viruses, prokaryotes, and eukaryotes) can be used in accordance with the present invention to provide herpesvirus nucleotide sequences (eg, DNA).
In particular to provide protection against a wide range of diseases. And many such genes are already known in the art. While not limited to any particular foreign DNA sequence, typically the foreign DNA sequence of interest is derived from a pathogen that causes a disease in birds that has an economic impact on the poultry industry. This gene may be derived from the organism in which the existing vaccine resides and vectorized (vector
Due to the novel advantages of the (ring) technique, this herpesvirus-derived vaccine is superior. Also, the gene of interest may be from a pathogen for which there is currently no vaccine but disease control is required. Typically, the gene of interest encodes an immunogenic polypeptide of the pathogen and may represent surface, secreted and structural proteins.

A related avian pathogen that is a target for herpesvirus vectorization is infectious laryngotracheitis virus (ILTV). ILTV is a member of the family Herpesviridae, and this pathogen causes an acute disease in chickens characterized by hypopnea, infectious bronchitis, and infiltration of open exudates.

Another target of the herpesvirus vectorization approach is Newcastle disease. Newcastle disease is a highly contagious and debilitating infectious disease, caused by Newcastle disease virus (NDV). NDV is a single-stranded RNA virus of the Paramyxoviridae family. Different pathogenic types of NDV (short latency,
(Pathogenic, xenogenic) vary with respect to the severity, specificity, and symptoms of the disease, but most types appear to infect the respiratory and nervous systems. NDV
The family infect chickens, turkeys and other bird species.

The present invention is also not limited to the use of a particular DNA sequence from an organism. Often, the selection of a foreign DNA sequence for insertion into the herpesvirus genome is based on the protein it encodes. Preferably, the foreign DNA sequence encodes an immunogenic polypeptide. Preferred immunogenic polypeptides expressed by the viral system of the present invention include a full-length (or near full-length) sequence encoding an antigen. Alternatively, shorter sequences that are immunogenic (ie, encode one or more epitopes) can be used. Shorter sequences can encode a neutralizing epitope, which is defined as an epitope that can elicit antibodies that neutralize viral infectivity in an in vitro assay. Preferably, the peptide should encode a protective epitope capable of eliciting a protective immune response in the host (ie, an antibody- and / or cell-mediated immune response that protects the immunized host from infection). . In some cases, the gene for a particular antigen may contain multiple introns or may be derived from an RNA virus, in which case the complementary DNA
Copies (cDNA) can be used.

It is also possible that only fragments of the nucleotide sequence of the gene can be used, rather than the complete sequence as found in a wild-type organism (these are sufficient to generate a protective immune response) Case). Where available, synthetic genes or fragments thereof may also be used. However, the present invention can be used with a wide variety of genes, fragments, etc., and is not limited to the genes, fragments, etc. described herein.

Thus, the antigen encoded by the foreign DNA sequence used in the present invention can be either a native or recombinant immunogenic polypeptide or fragment. They can be partial sequences, full-length sequences, or even fusions (eg, having a suitable leader sequence in a recombinant host or having additional antigenic sequences for another pathogen).

[0064] In a preferred embodiment, the mutant viruses and viral vectors of the invention are replicable. Thus, deletions from and / or insertions into the herpesvirus genome do not destroy the replication capacity of the herpesvirus. However, if the deletion and / or insertion disrupts or substantially inhibits the replication capacity of the herpes virus, the invention constructs an expression cassette comprising the herpes virus of the invention and transforms host cells with the cassette. DNA that has been deleted or destroyed
Includes the use of recombinant cell lines by providing a cell line or culture that expresses the protein encoded by the sequence.

These recombinant cell lines are capable of allowing a recombinant herpesvirus that is not replication competent to replicate and express a desired foreign DNA sequence or a fragment thereof encoded within the recombinant herpesvirus. . These cell lines are also very useful for producing recombinant herpesvirus by in vivo recombination, followed by DNA-mediated co-transfection.

When the methods and compositions of the present invention are used for vaccination, it is not limited to any particular administration. One example is parenteral administration. When administered parenterally, the vaccine may involve the use of a vaccine carrier. Vaccine carriers are well known in the art: for example, bovine serum albumin (BSA), human serum albumin (H
AS) and keyhole limpet hemocyanin (HLH). A preferred carrier protein, rotavirus VP6, is disclosed in EP-A-0259149.

The vaccine can also be administered orally in a suitable oral carrier, such as an enteric coated dosage form. Oral formulations include commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like. The oral vaccine composition comprises
It may take the form of a solution, suspension, tablet, pill, capsule, sustained release formulation or powder, containing from about 10% to about 95% active ingredient, preferably from about 25% to about 70% active ingredient . Oral vaccines may be preferred for eliciting mucosal immunity in combination with systemic immunity, which plays an important role in protection against gastrointestinal infections of pathogens.

In addition, vaccines are formulated into suppositories. For suppositories, the vaccine compositions will include conventional binders and carriers, such as polyalkaline glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w / w), preferably about 1% to about 2%.

[0069] Protocols for administering the vaccine compositions of the present invention to animals are within the skill of the art in light of the present disclosure. One of skill in the art will select a concentration of the vaccine composition at a dose effective to elicit an antibody and / or T-cell mediated immune response against the immunogenic fragment. To a large extent, this dosage is not considered important.

Typically, a vaccine composition will comprise a convenient volume of vehicle (eg, about 1-10
cc) in a manner that delivers between about 1 to about 1000 μg of the subunit antigen. Preferably, the dosage in a single immunization will deliver about 1 to about 500 μg of the subunit antigen, more preferably about 5-10 to about 100-200 μg (eg, 5-200 μg) of the subunit antigen.

The time of administration can also be important. For example, a primary inoculation can preferably be followed by a subsequent booster inoculation, if necessary. Optionally, it may also be preferable to administer a second booster immunization to the animals weeks to months after the first immunization. To ensure that a high level of protection against disease is maintained, it may be beneficial to re-administer the animals with booster immunity at regular intervals (eg, once every few years). Alternatively, the first dose may be administered orally, followed by a later inoculation, or vice versa. A preferred vaccination protocol is
It can be established through experimentation with conventional vaccination protocols.

The recombinant herpesvirus of the invention also provides a method of differentiating animals vaccinated by the vaccine of the invention from animals infected by a naturally occurring wild-type infectious herpesvirus or other pathogen. I can do it. This is possible because the recombinant herpes virus contains foreign DNA encoding a limited number of antigens from the above viruses that are required to confer protective immunity against the corresponding pathogen. As a result, host animals vaccinated with these recombinant herpesviruses will have the wild-type infectious Farbikius disease virus (bursal disease).
virus, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus or infectious bronchitis virus, can be distinguished by the absence of antigens normally present in wild-type virus. In addition, if the herpes virus vector contains a deletion of a portion of its genome that encodes an immunogenic polypeptide, the immune response from the vaccinated animal to the product of the deleted portion will be lost. Indicates the vaccinated animal.

The present invention also provides an animal in need of gene therapy to control gene deficiency,
A method for providing gene therapy, comprising the steps of: providing a live recombinant herpes virus, comprising a foreign nucleotide sequence encoding the gene in a non-deleted form, to a recombinant viral vector; Administering the genome under the conditions that allow the expression of the gene of interest in a target organ or tissue, whether the genome is integrated into the genome of the mammal or independently maintained extrachromosomally. Include.

[0074] These types of techniques for replacing deleted genes or portions thereof are used by those skilled in the art. See, for example, Anderson et al. (US Pat. No. 5,399,34).
No. 6) describes a technique relating to gene therapy. Furthermore, examples of nucleotide sequences of foreign DNA sequences, or portions thereof, that can be incorporated for use in convenient gene therapy include the cystic fibrosis transmembrane regulatory gene (CFTR gene (cyst
ic fibrosis transmembrane conductor
e regulator gene)), human mini-dystrophin gene, α-
Anti-trypsin gene and the like.

[0075] Methods for the construction, selection and purification of recombinant herpesviruses are detailed in the Examples below. The following examples serve to illustrate certain preferred embodiments and aspects of the invention and should not be construed as limiting the scope of the invention.

Examples Example 1 Preparation of Marek's Disease Virus (MDV-1) Stock Marek's Disease Virus stock sample was 2 mM glutamine, 100 units /
HAM 'containing ml penicillin, 100 units / ml streptomycin
1: 1 mixture of SF10 and 199 media (these components were obtained from Sigma or equivalent supplier and are referred to hereinafter as complete DME media) (1
% Fetal bovine serum) at a multiplicity of infection of 0.01 PFU / cell. After the cytopathic effect was completed, the media and cells were harvested, and the cells were pelleted in a clinical centrifuge at 3000 rpm for 5 minutes. Infected cells were resuspended in complete medium containing 20% fetal calf serum, 10% DMSO and stored frozen at -70 ° C.

Example 2 Preparation of Marek's Disease Virus (MDV-1) DNA All manipulations of Marek's disease virus were performed using strain GA5 (ATCC # 624) or Rispens CVI-988 (Vineland Labs). Was. For the preparation of MDV viral DNA from the cytoplasm of infected cells, primary chicken embryo fibroblasts were infected prior to cell overgrowth at an MOI sufficient to cause extensive cytopathic effects. All incubations were performed at 39 ° C. in a humidified incubator in 5% CO ₂ air. The highest DNA yields were obtained by collecting monolayers that were maximally infected but showed incomplete cell lysis (typically 5-7 days). Infected cells were collected by scraping the cells into the medium using a cell scraper. This cell suspension was placed in a GS-3 rotor for 3 hours.
Centrifuged at 5,000 rpm for 10 minutes at 5 ° C.

The resulting pellet was resuspended in cold PBS (20 ml / rotor bottle) and subjected to further centrifugation at 3000 rpm for 10 minutes with cooling. After decanting the PBS, the cell pellet was washed with RSB buffer (10 mM Tris pH
7.5, 1 mM EDTA and 1.5 mM MgCl ₂ ) in a 4 ml / rotor bottle. NP40 (Nonidet P-40; Sigma) was added to the sample to a final concentration of 0.5% with occasional mixing. The sample is centrifuged at 3000 rpm for 10 minutes with cooling to pellet the nuclei,
The cell debris was then removed. The supernatant fluid was carefully transferred to a 15 ml Corex centrifuge tube. Both EDTA (0.5 M pH 8.0) and SDS (sodium dodecyl sulfate; 20% storage) were added to the samples to final concentrations of 5 mM and 1%, respectively. 100 μl of proteinase-K (10 mg
/ Ml; Boehringer Mannheim) was added per 4 ml sample, mixed and incubated at 45 ° C for 1-2 hours. After this period, an equal volume of saturated phenol was added to the sample and mixed gently by hand. The sample was spun at 3000 rpm for 5 minutes in a clinical centrifuge to separate the phases. Sodium acetate (stock solution is 3M pH 5.2) at a final concentration of 0.3M is added to the aqueous phase and the nucleic acid is added after adding 2.5 volumes of cold absolute ethanol-
Precipitated at 70 ° C. for 30 minutes. The DNA in the sample was stored at 5 ° C in an HB-4 rotor.
And pelletized by spinning at 8000 rpm for 20 minutes. The supernatant was carefully removed and the DNA pellet was washed once with 25 ml of 80% ethanol. Dry the DNA pellet briefly by vacuum (2-3 minutes)
And 50 μl / TE buffer (10 mM) of rotor bottles of infected cells
Tris pH 7.5, 1 mM EDTA). Typically, viral DNA yields ranged between 5-10 μg / rotor bottle of infected cells. All viral DNA was stored at about 10 ° C.

Example 3 DNA Sequencing DNA sequencing was performed using ABI PRISM Dye Terminator C
Cycle Sequencing Ready Reaction Kit and Amplitaq DNA polymerase, FS (Perkin-Elmer;
According to the manufacturer's instructions) and perform the Perkin-Elmer / Applied B according to the manufacturer's instructions.
This was performed by electrophoresis on an iosystems automated DNA sequencer Model 373A. Reactions with both dGTP and dITP mixtures were performed to define areas of compression. Alternatively, the compression zone was separated on a formamide gel. The template is a double-stranded plasmid subclone or a single-stranded M13 subclone, and primers are generated either on the vector just outside the insert to be sequenced, or on a previously obtained sequence. did. The resulting sequences were collected and compared using DNAStar software.

Example 4 Molecular Biological Techniques Techniques for Manipulating Bacteria and DNA (Digestion with Restriction Endonucleases, Gel Electrophoresis, Extraction of DNA from Gels, Ligation, Phosphorylation Using Kinases) ,
Procedures, such as treatment with phosphatases, growing bacterial cultures, transforming bacteria with DNA, and other molecular biological methods). Sambrook
k, et al., Molecular Cloning A Laboratory Ma.
natural 2nd edition, Cold Spring Harbor Press, 19
89 and Current Protocols in Molecular
Biology (1992) John Wiley &Son's, Inc.
Described by Except as noted, they were used with minor modifications.

(Example 5) (Polymerase fill-in reaction) DNA was treated with 50 mM Tris pH 7.4, 50 mM KCl, and 5 mM.
Resuspended in buffer containing MgCl ₂ and 400 μM of each of the four deoxynucleotides. Ten units of Klenow DNA polymerase (BRL) were added and the reaction was allowed to proceed for 15 minutes at room temperature. The DNA was then phenol extracted and ethanol precipitated as described above.

Example 6 Cloning Using Polymerase Chain Reaction Using the polymerase chain reaction (PCR), simple restriction sites were introduced for various DNA manipulations. The procedure used is described in M.E. A. Innis et al. (PCR Prot
ocols A Guide to Methods and Applica
tions, 84-91, Academic Press, Inc. , San
Diego, 1990). Generally, amplified fragments were less than 500 base pairs in size, and critical regions of the amplified fragments were confirmed by DNA sequencing. The primer used in each case was
This is described in detail in the following description of the construction of the homology vector.

Example 7 Preparation of Lysate of Infected Cells Secondary in a 25 cm ² flask or 60 mm Petri dish
) A confluent monolayer of chicken embryo fibroblasts was infected with 100 μl of virus sample. After the cytopathic effect was complete, the media and cells were collected, and the cells were pelleted in a clinical centrifuge at 3000 rpm for 5 minutes. The cell pellet was resuspended in 250 μl of disruption buffer (2% sodium dodecyl sulfate, 2% β-mercaptoethanol). This sample
Sonicated on ice for 30 seconds and stored at -20 ° C.

Example 8 Western Blot Procedure Lysate samples and protein standards were prepared as described in Laemnli, U.S.A. K. (
1970) run on a polyacrylamide gel according to the procedure of Nature 277: 680. After electrophoresis of the gel, the protein was removed from Sambrook.
Transferred and processed according to (1989). The primary antibody is T
ris- sodium chloride and sodium azide (TSA: 1l of H ₂ O per 6.61g Tris-HCl, 0.97g Tris base, 9.0GNaCl
And 1: 5 dilution with 5% nonfat dry milk in 2.0 g sodium azide). The secondary antibody was conjugated to alkaline phosphatase and diluted 1: 1000 with TSA.

Example 9 cDNA Cloning Procedure cDNA cloning refers to the method used to convert RNA molecules into DNA molecules according to state of the art procedures. Applicants' method is described in (U. Gubler
And B. J Hoffman, Gene 25, 263-269). Bethesda Research Laboratories (Gai
thersburg, Md. ) Is very similar to the procedure used by the applicant, and contains a set of reagents and procedures that can be used to reproduce our results.
An NA cloning kit was designed.

For the cloning of viral mRNA species, host cell lines susceptible to infection by the virus were infected at 5-10 plaque forming units per cell. If cytopathic effects are evident but before any disruption, the medium is removed and the cells are
l lysis buffer (4 M guanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0, 0
. 5% N-lauryl sarcosine, 0.1 M β-mercaptoethanol). The cell lysate was poured into a sterile Dounce homogenizer and homogenized on ice for 8-10 minutes until the solution was homogeneous. For RNA purification, 8 ml of cell lysate was placed in a Beckman SW41 centrifuge tube at 3
. Gently overlaid 5 ml of CsCl solution (5.7 M CsCl, 25 mM sodium citrate pH 7.0). Samples were taken from Beckman SW41
The mixture was centrifuged in a rotor at 20 ° C. and 36000 rpm for 18 hours. The tube is placed on ice, and the supernatant from the tube is carefully removed by aspiration, RN
The A pellet was not disturbed. The pellet was resuspended in 400 μl of glass distilled water and 2.6 ml of a guanidine solution (7.5 M guanidine-HCL,
25 mM sodium citrate pH 7.0, 5 mM dithiothreitol). 0.37 volumes of 1 M acetic acid were added, followed by 0.75 volumes of cold ethanol, and the samples were placed at -20 ° C for 18 hours to precipitate RNA.
The precipitate was placed in an SS34 rotor in a Sorvall centrifuge for 10 minutes at 4 ° C.
Collected by centrifugation at 10,000 rpm. 1.0 pellet
Dissolved in ml of sterile water, centrifuged again at 13000 rpm and saved supernatant. RNA was re-extracted from the pellet twice more with 0.5 ml of sterile water as above and the supernatant was pooled. 0.1 volume of 2M potassium acetate solution was added to the sample, followed by 2 × cold ethanol, and the sample was placed at -20 ° C. for 18 hours. The precipitated RNA was placed in an SS34 rotor at 40 ° C.
Collected by centrifugation at 10,000 rpm for 10 minutes. The pellet was dissolved in 1 ml of sterile water and the concentration was obtained by absorption of A260 / 280. This RNA was stored at -70 ° C.

The mRNA containing the polyadenylation tail (poly-A) was selected using oligo dT cellulose (Pharmacia # 27 5543-0). 3 mg of total RNA is boiled and cooled and the binding buffer (0.1 M Tris pH 7
. 5, 0.5 M LiCl, 5 mM EDTA pH 8.0, 0.1% lithium dodecyl sulfate) applied to a 100 mg oligo dT cellulose column. The retained poly-A RNA was added to an elution buffer (5 mM Tris pH 7.5,
The column was eluted with 1 mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate. This mRNA was re-applied to the oligo dT column in binding buffer and eluted again with elution buffer. The sample was precipitated with 200 mM sodium acetate and 2 volumes of cold ethanol at -20 ° C for 18 hours. This RNA was resuspended in 50 μl of sterile water.

10 μg of poly-A RNA was added in 20 mM methylmercury hydroxide for 6 minutes
Denatured at 2 ° C. β-Mercaptoethanol was added to 75 mM and the samples were incubated for 5 minutes at 22 ° C. 0.25 ml of the reaction mixture for the first strand cDNA synthesis contained 1 μg oligo-dT primer (P-L
Biochemicals) or 1 μg of synthetic primer, 28 units of placental ribonuclease inhibitor (Bethesda Research L)
abs # 5518SA), 100 mM Tris pH 8.3, 140 mM
KCl, 10 mM MgCl ₂ , 0.8 mM DATP, dCTP, dGTP and dTTP (Pharmacia), 100 microcuries of ³² P-labeled dCTP (New England Nuclear # NEG-013H) and 180 units of AMV reverse transcriptase (Molecule)
ar Genetics Resources #MG 101). The reaction was incubated at 42 ° C. for 90 minutes, then 20 mM EDTA p
Termination was performed using H8.0. The sample was extracted with an equal volume of phenol / chloroform (1: 1) and precipitated at −20 ° C. for 3 hours with 2M ammonium acetate and 2 volumes of cold ethanol. After precipitation and centrifugation, the pellet was dissolved in 100 μl of sterile water. Samples are buffered (100 mM
Tris pH 7.5, 1 mM EDTA pH 8.0, 100 mM N
a-100) in a 15 ml G-100 Sephadex column (Pharmac).
ia) loaded on top. The rising of the extracted DNA fraction (leading
edge)) and the DNA was concentrated by lyophilization to a volume of about 100 μl, then the DNA was precipitated with ammonium acetate and ethanol as described above.

The entire first strand sample was treated with 50 μg / ml dNTPs and 5.4 units of DN.
A polymerase I (Boerchinger Mannheim # 642-71
1), and 100 units / ml coli DNA ligase (New
England Biolabs # 205) (total volume 50 μl) was used for the second strand reaction according to the method of Gubler and Hoffman (supra) except that it was used. After second strand synthesis, the cDNA was phenol / chloroform extracted,
And allowed to settle. The DNA was resuspended in 10 μl of sterile water and 1 μg RNase
A for 10 minutes at 22 ° C. and agarose gel (Sigma Type II agarose) in 40 mM Tris-acetate pH 6.85
Electrophoresis. The gel was stained with ethidium bromide, and DNA in the expected size range was excised from the gel and 8 mM Tris-
Electroeluted in acetate pH 6.85. About 100 electroeluted DNA
Lyophilized to μl and precipitated as above with ammonium acetate and ethanol. This DNA was resuspended in 20 μl of water.

An oligo dC tail was added to the DNA to facilitate cloning. The reaction was performed using the DNA, 100 mM calcium cacodylate (pH 7.2).
0.2 mM dithiothreitol, 2 mM CaCl ₂ , 80 μmol dC
TP, and 25 units of terminal deoxynucleotidyl transferase (Mo
linear Genetic Resources # S1001) to 5
Contained in 0 μl. After 30 minutes at 37 ° C., the reaction was terminated with 10 mM EDTA, and the sample was phenol / chloroform extracted and precipitated as described above.

The dC-tailed DNA sample was prepared by adding 200 μl of 0.01 M Tris pH7.
. 5, oligo dG tail (Bethesda Research Labs # 5355 SA / S) in 0.1 M NaCl, 1 mM EDTA pH 8.0.
B) into 200 ng plasmid vector pBR322 at 65 ° C. for 2 minutes,
Then, annealing was performed at 57 ° C. for 2 hours. D. Hanahan, Molec
urological Biology 166, 557-580, 1983, fresh competent E. coli. E. coli DH-1 cells were prepared and
Half of this annealed cDNA sample was used to transform in 20 00 μl cell aliquots. Transformed cells were placed on L-broth agar plates containing 10 μg / ml tetracycline. Colonies are picked from Ampscr
een (Bethesda Research Labs # 5537 UA)
Was used to screen for the presence of the insert into the ampicillin gene, and positive colonies were picked for analysis.

Example 10 DNA Transfection to Generate Recombinant Marek's Disease Virus This method is based on the following modifications of Kawai and Nishizawa, M.
ol. and Cell. Biol. 4: 1172-1174 (1984) based on the polybrene-DMSO procedure. The generation of recombinant MDV virus is accomplished by combining MDV viral DNA with the desired foreign D
It relies on homologous recombination with a plasmid homology vector containing NA. Transfections were performed on 6 cm plates (Corning plastic) of 50% confluent primary chicken embryo fibroblast (CFF) cells. The cells were grown in CEF growth medium (1 × F10 / 199, 5% fetal bovine serum, 2% glutamine, 1% non-essential amino acids, and 2%) containing 4 μg / ml polybrene (4 mg / ml stock in 1 × HBSS). % Penicillin / streptomycin). For co-transfection into CEF cells, 5 μg of intact MDV DNA was added to 30 μg / ml polybrene (4 mg / l in 1 × HBSS).
(ml stock) in 1 ml CEF medium. Then, this DNA
-Polybrene suspension (1 ml) was added to a 6 cm plate of CEF cells with aspirated medium and incubated at 39 ° C for 30 minutes. The plate was rocked periodically during this time to redistribute the inoculum. After this period, 4 ml of C
EF growth media was added directly to wash the plates and incubated at 39 ° C. for an additional 2.5 hours. At this point, the medium was removed from each plate and the cells were added to 30% DMSO in 1 × HBSS (dimethyl sulfoxide, J
. T. Baker Chemical Co. , Phillipsburg, N
J) Shocked with 2 ml for 4 minutes at room temperature. The 30% DMSO was carefully removed and the monolayer was washed once with 1 × HBSS at room temperature. The cells were then incubated at 39 ° C. after adding 5 ml of CEF growth medium to the cells. The next day, the medium was changed to remove the last residue of DMSO and stimulated cell growth. The cytopathic effect from the virus becomes apparent within 6 days. Production of high titer stocks (80% -90% CPE) can usually take place within a week from this date. An MDV stock sample was prepared using 20% fetal calf serum,
Prepared by resuspending infected cells in CEF growth medium with 10% DMSO and stored at -70 ° C.

Example 11 Screening for Recombinant Marek's Disease Virus Expressing β-Galactosidase (Bluogal and CPRC Assays) or Screening for Recombinant Marek's Disease Virus Expressing β-Glucuronidase (X-Gluc
Assay)) When the E. coli β-galactosidase (lacZ) marker gene was incorporated into the recombinant virus, plaques containing the recombinant were visualized by one of two simple methods. In the first method, the chemical Bluogal ^™ (Life Sciences Technology, Bethesda, M
D) was incorporated into the agarose overlay during the plaque assay (200 μg /
ml). The plaques expressing active β-galactosidase turned blue. The blue plaque was then picked into fresh CEF cells and purified by further blue plaque isolation. In the second method, CPRG (Boehr
(inner Mannheim) was incorporated into the agarose overlay during the plaque assay (400 μg / ml). Plaques expressing active β-galactosidase turned red. The red plaque was then picked into fresh CEF cells and purified by further red plaque isolation. In both cases, the virus was typically purified by three to four plaque purifications.

E. When the E. coli β-glucuronidase (uidA) marker gene was incorporated into the recombinant virus, the plaque containing the recombinant virus was transformed into a chromogenic substrate X
-Β-D-gluUA CHX (X-GLUC; 5-bromo-4-chloro-3-
Indoxyl-β-D-glucuronic acid, cyclohexylammonium salt; Bio
synth AG (Switzerland) was incorporated into the agarose overlay during the plaque assay (200 μg / ml). Plaques expressing active β-glucuronidase turned blue. The blue plaque was then picked into fresh CEF cells and purified by further blue plaque isolation.

Example 12 Exogenous D in Recombinant Marek's Disease Virus Using Black Plaque Assay
Screening for NA Sequence Expression To analyze the expression of foreign antigens expressed by recombinant MDV virus,
Infect monolayers of EF cells with recombinant MDV, overlay nutrient agarose medium and incubate at 39 ° C for 4-5 days. Once the plaque has developed, the agarose overlay is removed from the dish and the monolayer is rinsed with 1 × PBS and incubated at room temperature for 10 minutes.
Fix with 100% methanol for minutes and air dry cells. After rehydration of the plate with PBS, the primary antibody is diluted with PBS to the appropriate dilution and added at room temperature.
Incubate with the cell monolayer for hours to overnight. The unbound antibody is then
Remove from cells by three washes with BS at room temperature. The alkaline phosphatase-conjugated secondary antibody is diluted in PBS and incubated with the cells for 2 hours at room temperature. Unbound secondary antibody is then removed by washing the cells three times with PBS at room temperature. The monolayer was then washed with a color buffer (100 mM
Rinse in Tris pH 9.5 / 100 mM NaCl / 5 mM MgCl ₂ ) and then freshly prepared substrate solution (0.3 mg / ml N 2 in color development buffer)
(intro Blue tetrazolium + 0.15 mg / ml 5-bromo-4-chloro-3-indolyl phosphatase) for 10 minutes to overnight at room temperature. Finally, the reaction was carried out using TE (10 mM Tris, pH
Stop by replacing with 7.5 / 1 mM EDTA). Plaques expressing the correct antigen stain black.

Example 13 Plasmid with Foreign DNA Inserted in Open Reading Frame UL54.5 of Marek's Disease Virus Type 1 Plasmid 440-29.2 was introduced into Marek's disease virus type 1 (MDV-1) It was constructed for the purpose of inserting DNA. This plasmid contains an approximately 2596 base pair BamHI "M" genomic fragment of Marek's disease virus type 1 (SEQ ID NO: 1).
)including. The three open reading frames in this BamHI “M” fragment are the UL54 (ICP27) ORF (positions 1 to 1353 of SEQ ID NO: 1)
, An previously unidentified ORF (hence the name UL54.5) (SEQ ID NO: 1)
2187 to 1483) and UL55 (positions 2459 to 259 of SEQ ID NO: 1)
(No. 3) Herpesvirus homolog (see Tables 1 and 2). M
The DNA sequence (1492 base pairs) spanning the DV-1 UL54 (ICP27) gene has been published (Virology Oct 1994; 204 (1):
242-50). MDV ICP27 (based on significant similarity to HSV-1 ICP27) is 1419 nucleotides long and has 473 amino acids (5
4.5 kDa). The UL55 ORF is truncated and contains only the first 49 amino acids of the protein. A potential ORF located between UL5 and UL55 was identified. This ORF is 705 base pairs long and probably encodes a protein 235 amino acids in size. This UL54.
A similar gene was identified in MDV-2 by BLAST search of the protein database using the five amino acid sequence (Virology May 15, 1994; 201 (1): 142-6). It was noted that this MDV-2 gene shares low homology with the first open reading frame (ORF-1) of equine herpesvirus type 1. UL54. Derived from MDV-1 and MDV-2.
The similarity index of the 5 proteins is 6 over a consensus length of 170
3%. UL54.5 is transcribed in the opposite direction to UL54 and UL55.

[0097] The UL54.5 ORF is non-essential, and foreign DNA is inserted into or between the ORFs in the intergenic region. All restriction sites in this region are useful as insertion sites for foreign DNA. Restriction enzyme sites in this region that are not unique are those that convert that site into a unique restriction enzyme recognition sequence.
It is changed by inserting a linker. Preferably, the restriction enzyme site used for insertion of foreign DNA is the NdeI site at about nucleotide 2596 in this 2596 base pair BamHI "M" genomic fragment. This insertion site
In the L54.5 ORF is between amino acids 4 and 5 of this open reading frame. This plasmid vector is called pSP64 (Promega).
It was derived from the approximately 3045 base pair BamHI restriction fragment of a). Fragment 1 is an approximately 2596 base pair BamHI "M" fragment of Marek's disease virus type 1. Plasmid 440-29.2 was used to create a homology vector for insertion of foreign DNA into recombinant Marek's disease virus.

Example 14 Plasmid with Infectious Laryngotracheitis Virus DNA Inserted in Open Reading Frame UL54.5 of Marek's Disease Virus Type 1 Plasmid 980-85.1 was replaced with recombinant Marek's disease virus type 1 (MDV-1)
It was constructed for the purpose of inserting foreign DNA into it. This plasmid is MDV-
ILT virus gD and gI genes flanked by I.T. E. coli β-galactosidase (lacZ) marker gene is incorporated.
These genes were inserted into a unique NdeI site that was converted to a PacI site using a synthetic DNA linker. Upstream of this foreign DNA sequence is an approximately 422 base pair fragment of MDV DNA. Downstream of this foreign DNA sequence is MDV D
It is an approximately 2174 base pair fragment of NA. ILT virus gD and gI genes and E. coli. The direction of transcription of the E. coli β-galactosidase (lacZ) marker gene is opposite to the direction of transcription of the MDV UL54 ORF and UL55 ORF. This plasmid was used for DNA transfection to produce recombinant Marek's disease virus (Example 10), as well as screening for recombinant Marek's disease virus expressing β-galactosidase (Bluelog).
al and CPRC assays) or screening for recombinant Marek's disease virus expressing β-glucuronidase (X-Gluc assay) (Example 1).
When used according to 1), a virus is generated which contains DNA encoding the foreign DNA sequence. The ILTV gD and gI genes are expressed as overlapping transcripts from each of their own endogenous ILTV promoters, and share their own endogenous polyadenylation signals, and The E. coli β-galactosidase (lacZ) marker gene is transcribed from the PRV gX promoter, followed by a PRV gX polyadenylation signal.

Plasmid 980-85.1 was constructed using standard recombinant DNA techniques by combining restriction fragments from the following sources with the synthetic DNA sequence. ILT gD, gI, and E. g. coli β-galactosidase (la
cZ) The marker gene was inserted as a cassette into the homology vector 440-29.2 at the unique NdeI site converted to a PacI site using a synthetic DNA linker. This plasmid vector contains about 3 of pSP64 (Promega).
It was derived from a 045 base pair HindIII restriction fragment. Fragment 1 is an approximately 418 base pair BamHI-N of the MDV BamHI restriction fragment M.
This is a deI restriction subfragment. Fragment 2 contains ILT Asp 71
Approximately 3556 base pairs of SalI of 81 genomic fragment # 8 (10.6 kb)
~ HindIII restriction subfragment. Fragment 3 contains PRV B
This is an approximately 413 base pair SalI to BamHI restriction subfragment of the amHI restriction fragment # 10. Fragment 4 was obtained from plasmid pJF751 (11
) Is an approximately 3010 base pair BamHI to PvuII restriction subfragment. Fragment 5 is approximately 754 of PRV BamHI genomic fragment # 7.
Base pair NdeI to SalI restriction subfragment. Fragment 6 is
About 2174 base pairs of NdeI to Ba of MDV BamHI restriction fragment M
mHI restriction subfragment.

Example 15 Plasmid in which Newcastle Disease Virus DNA was Inserted into Open Reading Frame UL54.5 of Marek's Disease Virus Type 1 Plasmid 980-46.74 was replaced with recombinant Marek's disease virus type 1 (MDV- 1
) Was constructed for the purpose of inserting foreign DNA into it. This plasmid is E. coli, flanked by MDV DNA. The E. coli β-galactosidase (lacZ) marker gene as well as the Newcastle disease virus (NDV) F gene are integrated. E
. E. coli β-galactosidase (lacZ) marker gene as well as the NDVF gene at the unique NdeI site converted to a PacI site using a synthetic DNA linker, and a cassette into the homology vector 440-29.2 (Example 13). As inserted.

Upstream of this foreign DAN sequence is an approximately 422 base pair fragment of MDV DNA. Downstream of this foreign DNA sequence is an approximately 2174 base pair fragment of MDV DNA. E. FIG. The direction of transcription of the E. coli β-galactosidase (lacZ) marker gene as well as the NDV F gene is opposite to the direction of transcription of the MDV UL54 and UL55 ORFs. This plasmid was transfected with DNA to generate recombinant Marek's disease virus (Example 10), and screened for recombinant Marek's disease virus expressing β-galactosidase (Bluogal and CPRC assays) or expressing β-glucuronidase. Screening for Recombinant Marek's Disease Virus (X-Glu
c) Assay (Example 11) results in a virus containing DNA encoding the foreign DNA sequence. The NDVF gene is under the control of the HCMV immediate early promoter, followed by the HSV TK polyadenylation signal. E
. The E. coli β-galactosidase (lacZ) marker gene was PRV g
Transcribed from the X promoter, followed by a PRV gX polyadenylation signal.

Plasmid 980-85.1 was constructed using standard recombinant DNA techniques by combining restriction fragments from the following sources with the synthetic DNA sequence. E. FIG. E. coli β-galactosidase (lacZ) marker gene as well as the NDVF gene at the unique NdeI site converted to a PacI site using a synthetic DNA linker, and a cassette into the homology vector 440-29.2 (Example 13). As inserted. This plasmid vector is called pSP64 (Prome
ga) derived from an approximately 3045 bp HindIII restriction fragment. Fragment 1 is an approximately 418 base pair B fragment of the MDV BamHI restriction fragment M.
amHI to NdeI restriction subfragment. Fragment 2 is the PRV
Approximately 413 base pairs of SalI to BamHI of BamHI restriction fragment # 10
Restriction subfragment. Fragment 3 is the plasmid pJF751
It is a BamHI to PvuII restriction fragment of about 3010 base pairs. Fragment 4 is an approximately 754 base pair Nd of PRV BamHI restriction fragment # 7.
eI to SalI restriction subfragment. Fragment 5 is an approximately 1191 base pair PstI to AvaII restriction subfragment of the XbaIE fragment of the HCMV genome. Fragment 6 is an approximately 1812 base pair BamHI to PstI restriction fragment of the full length NDV F cDNA clone (strain B1). Fragment 7 is approximately 78 of the HSV BamHI restriction fragment Q.
4 base pair SmaI to SmaI restriction subfragment. The last fragment is the approximately 2174 base pair Nd of the MDV BamHI restriction fragment "M".
El to BamHI restriction subfragments.

Example 16 Plasmid Having Foreign DNA Inserted in Open Reading Frame UL43 of Marek's Disease Virus Type 1 Plasmid 962-80.1 was introduced into Marek's disease virus type 1 (MDV-1) It was constructed for the purpose of inserting DNA. This is the type 1 Marek's disease virus B
Approximately 3212 base pairs of Sa contained within the amHI "B" genomic fragment
Includes the cI-BglII subfragment (SEQ ID NO: 2). 3212 base pairs of S
The three open reading frames within the acI to BglII subfragments are UL42 ORF (positions 35 to 1144 of SEQ ID NO: 2), UL43 (positions 1304 to 2566 of SEQ ID NO: 2), and UL44 (gC) (SEQ ID NO: 2 Of 27
(Positions 86-3220) (see FIGS. 3 and 4). The DNA sequence (732 base pairs) spanning the MDV-1 UL44 (gC) gene and promoter region has been published (Virus Genes).
3, 125-137 (1989)). The DNA sequence of the MDV-2 UL42, UL43 and UL44 genes has been published (J. Gen. Virol. 7).
9 (Pt 8), 1997-2001 (1998)). MDV-1 and MDV
The similarity index of the UL42 protein from -2 is 74 percent for a consensus length of 369 amino acids. UL43 from MDV-1 and MDV-2
The protein similarity index is 54 percent relative to a consensus length of 401 amino acids. The similarity index for the UL44 proteins from MDV-1 and MDV-2 is 45 percent for a consensus length of 145 amino acids. MD
The V-1 UL43 ORF is non-essential, and foreign DNA is transferred to this ORF.
Within or in the intergenic region between ORFs. Any restriction site within this region is useful as a foreign DNA insertion site. Restriction sites in this region that are not unique are altered by insertion of a DNA linker. The DNA linker converts this site into a unique restriction enzyme recognition sequence. Preferably, the foreign DN
The restriction enzyme site used for insertion of A is Marc type 1 virus BamHI "B
An XhoI site at approximately nucleotide 1386 in the 3212 base pair SacI to BglII subfragment contained within the genomic fragment. The insertion site is in the UL43 ORF between amino acids 29 and 30 of the open reading frame. This plasmid vector contains pSP64 (Pr
omega) from a BamHI restriction fragment of approximately 3045 base pairs. Fragment 1 is an approximately 3212 base pair SacI-BglII subfragment contained within the BamHI "B" genomic fragment of Marek's disease virus type 1. Plasmid 962-80.1 was used to create a homologous vector for insertion of foreign DNA into recombinant Marek's disease virus.

Example 17 Plasmid Having Infectious Laryngotracheitis Virus DNA Inserted in Open Reading Frame UL43 of Marek's Disease Virus Type 1 Plasmid 980-85.22 was replaced with recombinant Marek's disease virus type 1 ( MDV-1
) Was constructed for the purpose of inserting foreign DNA. This is the ILT virus gD
Gene and gI gene and E. coli. coli β-galactosidase (lac
Z) A marker gene has been integrated adjacent to MDV-1 DNA. These genes were converted to unique PacI sites using a synthetic DNA linker and the only X
Inserted at the hoI site. Upstream of the foreign DNA sequence is approximately 13 MDV DNA.
It is a fragment of 86 base pairs. Downstream of the foreign DNA sequence is an approximately 1826 base pair fragment of MDV DNA. ILT virus gD and gI genes and E. coli. The transcription direction of the E. coli β-galactosidase (lacZ) marker gene is the same as that of MDV UL42 and UL43 ORFs. This plasmid was used for DNA transfection to produce recombinant Marek's disease virus (Example 10) and screening for recombinant Marek's disease virus expressing β-galactosidase (Bluogal and Cprg assays), or expressing β-glucuronidase. When used according to the screening of recombinant Marek's disease virus (X-Gluc assay) (Example 11), foreign D
A virus is generated that contains DNA encoding the NA sequence. The ILTV gD and gI genes are expressed as overlapping transcripts from their own respective endogenous ILTV promoters and share their own endogenous polyadenylation signals, and The E. coli β-galactosidase (lacZ) marker gene is transcribed from the PRV gX promoter, followed by a PRV gX polyadenylation signal.

Plasmid 980-85.22 was constructed utilizing standard recombinant DNA techniques by joining restriction fragments from the following sources to the synthetic DNA sequence. ILT gD, gI, and E. g. coli β-galactosidase (l
The acZ) marker gene was inserted as a cassette into the homology vector 962-80.1 at a unique XhoI site, which was converted to a PacI site using a synthetic DNA linker. This plasmid vector is called pSP64 (Prom
(Ega, Madison, Wis.) from the HindIII restriction fragment of approximately 3045 base pairs. Fragment 1 is from the BamH type 1 Marek's disease virus
Approximately 1386 base pairs of SacI to I "B" genomic fragment
XhoI restriction subfragment. Fragment 2 contains ILTAsp718
Approximately 3556 of I genome fragment number 8 (10.6 kilobases)
Base pair SalI to HindIII restriction subfragment. Fragment 3 is an approximately 413 base pair SalI to BamHI restriction subfragment of PRV BamHI restriction fragment no. Fragment 4 is
Approximately 3010 base pairs BamHI to Pvu of plasmid pJF751
II restriction fragment (11). Fragment 5 is the PRV BamHI
It is an approximately 754 base pair NdeI to SalI restriction subfragment of restriction fragment number 7. Fragment 6 is from Bam of the Marek's disease virus type 1.
Approximately 1826 base pairs of XhoI contained within the HI "B" genomic fragment
〜BglII restriction subfragment.

Example 18 Recombinant Marek's Disease Virus Type 1 with Infectious Laryngotracheitis Virus DNA Inserted in Open Reading Frame UL43 S-MDV-006 expresses three foreign DNA sequences. Type Marek's disease virus. ILT virus gD and gI genes, and E. coli. coli β
-The galactosidase (lacZ) marker gene is replaced with a unique PacI restriction site (
UL43 OR of the approximately 3212 base pair SacI-BglII subfragment contained within the BamHI "B" genomic fragment of Marek's disease virus type 1
F) (PacI linker inserted into the unique XhoI restriction site in F). I
The LTV gD and gI genes have their own respective endogenous ILs.
Expressed as duplicate transcripts from the TV promoter, and share their own endogenous polyadenylation signal; coli β-galactosidase (
The lacZ) marker gene is transcribed from the PRV gX promoter, followed by a PRV gX polyadenylation signal. S-MDV-006 is S-MD
V-002 (MDV-1; CVI-988 Rispens). In a DNA transfection procedure to produce a recombinant Marek's disease virus (Example 10), the homology vector 980-85.22 and the virus S-MDV were used.
This was achieved utilizing -002. Co-transfection stock
Screened by β-glucuronidase (X-Gluc assay) (
Example 11). The end result of red plaque purification was a recombinant virus named S-MDV-006. This virus was expressed in β-galactosidase expression monitored by a blue plaque assay as described in Example 11,
The purity and the stability of the insert over multiple passages were assayed. After the first four rounds of purification, all plaques observed are blue, indicating that the virus is pure, stable, and expresses foreign DNA sequences.

S-MDV-006 was assayed for expression of ILT-specific antigen using a screen for expression of foreign DNA sequences in recombinant Marek's disease virus using a black plaque assay (Example 12). Polyclonal chicken anti-ILT serum (SPAFAS) was shown to react specifically with S-MDV-006 plaques and not specifically with S-MDV-002 negative control plaques. All S-MDV-006 observed plaques were
Reacts with polyclonal sera, indicating that the virus has
This indicates that the sequence was stably expressed. Assays described herein were performed using CE
Performed on F cells, which indicated that CEF cells were a suitable culture medium for the production of MDV recombinant vaccines.

S-MDV-006 is a recombinant Marek's disease virus type 1 that expresses ILT gD and gI proteins and is useful as a vaccine in ILT infection. S-MDV-006 is also an ILT gD protein and gI
Useful for protein expression.

Example 19 Plasmid Having Newcastle Disease Virus DNA Inserted in Open Reading Frame UL43 of Marek's Disease Virus Type 1 Plasmid 980-60.02 was replaced with recombinant Marek's disease virus type 1 (MDV-1)
) Was constructed for the purpose of inserting foreign DNA. This is E.I. coli β-
The galactosidase (lacZ) marker gene and the Newcastle disease virus (NDV) F gene have been integrated adjacent to MDV DNA. This E. c
oli β-galactosidase (lacZ) marker gene and the NDV F gene were the only Xhos converted to a PacI site using a synthetic DNA linker.
At the I site, it was inserted as a cassette into the homology vector 962-80.1.

[0110] Upstream of the foreign DNA sequence is an approximately 1386 base pair fragment of MDV DNA. Downstream of the foreign DNA sequence is an approximately 1826 base pair fragment of MDV DNA. E. FIG. The transcription direction of the E. coli β-galactosidase (lacZ) marker gene and the NDVF gene was determined by MDV UL42 and ULV.
43 Same transfer direction as ORF. This plasmid was used for DNA transfection to produce recombinant Marek's disease virus (Example 10) and screening for recombinant Marek's disease virus expressing β-galactosidase (Bluelog).
When used according to the al and Cprg assays) or screening for recombinant Marek's disease virus expressing β-glucuronidase (X-Gluc assay) (Example 11), a virus is generated that contains DNA encoding foreign DNA sequences. The NDVF gene is under the control of the HCMV immediate early promoter, followed by the HSV TK polyadenylation signal. E. FIG. An E. coli β-galactosidase (lacZ) marker gene was transcribed from the PRV gX promoter,
This is followed by a PRV gX polyadenylation signal.

Plasmid 980-85.1 was constructed utilizing standard recombinant DNA techniques by ligating restriction fragments from the following sources with the synthetic DNA sequence. E. FIG. The E. coli β-galactosidase (lacZ) marker gene and the NDV F gene were inserted as cassettes into the homology vector 962-80.1 at the unique XhoI site converted to a PacI site using a synthetic DNA linker. This plasmid vector contains approximately 30 of pSP64 (Promega).
It was derived from a 45 base pair HindIII restriction fragment.

Fragment 1 is a SacI to XhoI restriction subfragment of approximately 1386 base pairs contained within the BamHI “B” genomic fragment of Marek's disease virus type 1.

Fragment 2 is the PRV BamHI restriction fragment number 10
It is a SalI to BamHI restriction subfragment of approximately 413 base pairs. Fragment 3 is the approximately 3010 base pair Ba of plasmid pJF751.
mHI to PvuII restriction fragment. Fragment 4 is PRV Ba
Approximately 754 base pairs of NdeI to Sa of mHI restriction fragment number 7
This is an I I restriction subfragment. Fragment 5 contains the HCMV genome XbaI
It is a PstI to AvaII restriction subfragment of approximately 1191 base pairs of the E fragment. Fragment 6 is a BamHI to PstI restriction fragment of approximately 1812 base pairs of the full length NDV F cDNA clone (strain B1). Fragment 7 is an approximately 784 base pair SmaI to SmaI restriction subfragment of the HSV BamHI restriction fragment Q. The last fragment is an approximately 1826 base pair XhoI to BglII restriction subfragment contained within the BamHI "B" genomic fragment of Marek's disease virus type 1.

Example 20 Recombinant Marek's Disease Virus Type 1 with Newcastle Disease Virus DNA Inserted in the Open Reading Frame UL43 S-MDV-004 is a type 1 Marek's disease expressing two foreign DNA sequences It is a virus. The gene for the Newcastle disease virus fusion (F); col
i.beta.-galactosidase (lacZ) marker gene is ligated to a unique PacI restriction site (UL43 of the approximately 3212 base pair SacI-BglII subfragment contained within the BamHI "B" genomic fragment of Marek's disease virus type 1).
(PacI linker inserted into the unique XhoI restriction site in the ORF). This NDVF gene is under the control of the HCMV immediate early promoter and coli β-galactosidase (lacZ) marker gene
Transcribed from the gX promoter and followed by a PRV gX polyadenylation signal. S-MDV-004 is S-MDV-002 (MDV-1; CVI-9
88 Rispens). In the DNA transfection to produce the recombinant Marek's disease virus (Example 10), the homology vector 980-6 was used.
This is accomplished utilizing 0.02 and the virus S-MDV-002. Co-transfection stocks were screened for recombinant Marek's disease virus expressing β-galactosidase (Bluogal and Cprg assays) or for recombinant Marek's disease virus expressing β-glucuronidase (X-Gluc assay) (Example 11). ). The end result of the red plaque purification was a recombinant virus named S-MDV-004. The virus is assayed for β-galactosidase expression, purity, and insert stability over multiple passages monitored by a blue plaque assay as described in Example 11. After the first four rounds of purification, all plaques observed are blue, indicating that the virus is pure, stable, and expresses foreign DNA sequences.

S-MDV-004 is assayed for NDV-specific antigen expression using a screen for expression of foreign DNA sequences in recombinant Marek's disease virus using a black plaque assay (Example 12). Monoclonal antibodies specific for NDVF are shown to react specifically with S-MDV-004 plaques and not specifically with S-MDV-002 negative control plaques. All S-MDV-004 observed plaques reacted with polyclonal serum, indicating that the virus is stably expressing the NDV F gene. The assays described herein were performed on CEF cells, which indicates that CEF cells are a suitable culture medium for the production of MDV recombinant vaccine.

S-MDV-004 is a recombinant Marek's disease virus type 1 that expresses the NDV F protein and is useful as a vaccine in NDV infection. S-MDV-
004 is also useful for F protein expression.

Example 21 (Open reading frame UL7 of Marek's disease virus type 1 and / or
Alternatively, a plasmid having foreign DNA inserted between open reading frames UL8 and UL7) A plasmid is constructed for the purpose of inserting foreign DNA into Marek's disease virus type 1 (MDV-1). It contains an approximately 7316 base pair subfragment (SEQ ID NO: 3) contained within the BamHI "G" genomic fragment of Marek's disease virus type 1. The five open reading frames within the 7316 base pair subfragment are UL9 ORF (positions 1-425 of SEQ ID NO: 3), UL8
(Positions 439 to 2748 of SEQ ID NO: 3), UL7 (gC) (369 of SEQ ID NO: 3)
Herpesvirus homologues of positions 9 to 2782), UL6 (positions 5704 to 3536 of SEQ ID NO: 3), and UL5 (positions 5772 to 7316 of SEQ ID NO: 3) (see FIGS. 5 and 6). . The region between ORF UL8 and UL7 (positions 2749-2781 of SEQ ID NO: 3) and the portion of OFR7 that does not overlap with ORF UL6 (positions 2782-3535 of SEQ ID NO: 3) is not essential for virus replication, It can then be used to generate mutant and / or recombinant viruses.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided for purposes of illustration only, and the present invention is to be construed with the full scope of equivalents to which such claims are entitled. It should be limited only by scope.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a BamHI restriction map of the MDV genome, specifically indicating the location of the “M” fragment.

FIG. 2 is a map showing the open reading frame in the BamHI “M” fragment of the MDV genome.

FIG. 3 is a BamHI restriction map of the MDV genome, in particular, SacI-Bg
The position of the III fragment is indicated.

FIG. 4 is a map showing the open reading frame in the SacI-BglIII fragment of the MDV genome.

FIG. 5 is a BamHI restriction map of the MDV genome, specifically showing the location of the “G” fragment.

FIG. 6 is a map of the open reading frame in the BamHI “G” fragment of the MDV genome.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/265 A61K 48/00 48/00 A61P 31/12 171 A61P 31/12 171 C12R 1:93 (C12N 7/00 C12N 15/00 ZNAA C12R 1:93) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, A L, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE , HR, HU, ID, IL, IN, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LU, LV, MA, MD, MG, MK, MN, MX, NO, NZ, PL, PT, RO, RU, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, US, UZ, VN, YU, ZA F term (reference) 4B024 AA10 BA32 DA02 EA02 FA02 GA11 HA01 HA20 4B065 AA95X AA95Y AB01 AC20 BA01 CA43 4C084 AA02 AA03 AA13 AA17 BA44 CA01 CA53 MA17 MA22 MA23 MA31 MA35 MA36 MA37 MA41 MA43 MA52 MA55 MA58 MA59 MA66 NA14 ZB332 ZC612 4C085 AA03 BB08 BA81GG81 BA81 GG81 DD81 AA01 AA02 BC83 CA09 CA12 MA17 MA22 MA23 MA31 MA35 MA36 MA37 MA41 MA43 MA52 MA58 MA59 MA66 NA14 ZB33 ZC61

Claims (20)

[Claims] 1. A recombinant avian herpesvirus comprising a foreign DNA sequence inserted into the UL54.5 open reading frame of Marek's disease virus (MDV). 2. The recombinant herpesvirus according to claim 1, wherein said exogenous DNA encodes a polypeptide. 3. The recombinant herpesvirus of claim 2, wherein said polypeptide contains more than 10 amino acids. 4. The recombinant herpesvirus according to claim 2, wherein said polypeptide is antigenic. 5. The method of claim 1, wherein the avian herpes virus is a type I Marek's disease virus (MD).
The recombinant herpesvirus according to claim 1, comprising V-1). 6. The recombinant herpesvirus according to claim 5, wherein said exogenous DNA sequence is under the control of a herpesvirus promoter located upstream of said exogenous DNA sequence, and The foreign DNA sequence is PRV gX
A recombinant herpesvirus selected from the group consisting of a promoter, MDV gB promoter, MDV gA promoter, MDV gD promoter, ILTV gB promoter, ILTV gI promoter, HCMV immediate early promoter, and substantially homologous sequences. 7. The recombinant herpes virus according to claim 5, wherein said exogenous DNA sequence is chicken anemia virus, infectious Farbikius disease virus, Marek's disease virus, Newcastle disease virus, infectious laryngeal trachea. A recombinant herpes virus, which encodes an antigenic polypeptide derived from a virus selected from the group consisting of a flame virus, an infectious bronchitis virus, and a substantially homologous sequence; 8. The recombinant herpes virus according to claim 7, wherein said exogenous DNA sequence is selected from the group consisting of VP2, VP3, VP4, and substantially homologous sequences. A recombinant herpesvirus comprising a DNA sequence encoding an antigenic polypeptide from a disease virus. 9. The recombinant herpes virus according to claim 7, wherein said exogenous DNA is selected from the group consisting of B glycoprotein, D glycoprotein, A glycoprotein, and a substantially homologous sequence. A recombinant herpesvirus comprising a DNA sequence encoding an antigenic polypeptide from Marek's disease virus. 10. The recombinant herpes virus according to claim 7, wherein the exogenous DNA sequence is derived from Newcastle disease virus, wherein the exogenous DNA sequence is selected from the group consisting of F, HN, and a substantially homologous sequence. DN encoding antigenic polypeptide
A recombinant herpesvirus comprising the A sequence. 11. The recombinant herpesvirus of claim 7, wherein said exogenous DNA is selected from the group consisting of a spike protein, a nucleocapsid protein, a matrix protein, and a substantially homologous sequence. A recombinant herpesvirus comprising a DNA sequence encoding an antigenic polypeptide derived from sexual bronchitis virus. 12. A mutant avian herpesvirus comprising a deletion of at least a portion of the UL54.5 open reading frame of Marek's disease virus (MDV). 13. The mutant herpesvirus according to claim 12, wherein the UL54.5 open reading frame is completely deleted. 14. A recombinant avian herpesvirus comprising a foreign DNA sequence inserted into the UL43 open reading frame of Marek's disease virus. 15. The method of claim 1, wherein the exogenous DNA encodes a polypeptide.
4. The recombinant herpesvirus according to 4. 16. The recombinant herpesvirus of claim 15, wherein said polypeptide comprises more than 10 amino acids. 17. The recombinant herpesvirus according to claim 15, wherein said polypeptide is antigenic. 18. The herpes simplex virus according to claim 1, wherein the herpes simplex virus is a type I Marek's disease virus (MD).
18. The recombinant herpesvirus according to claim 17, comprising V-1). 19. The recombinant herpesvirus of claim 14, wherein said exogenous DNA sequence is under the control of a herpesvirus promoter located upstream of said exogenous DNA sequence, and The foreign DNA sequence is PRV
gX promoter, MDV gB promoter, MDV gA promoter, M
A recombinant herpesvirus selected from the group consisting of a DV gD promoter, an ILTV gB promoter, an ILTV gI promoter, an HCMV immediate early promoter, and a substantially homologous sequence. 20. The recombinant herpes virus according to claim 19, wherein said exogenous DNA sequence is chicken anemia virus, infectious Farbikius disease virus, Marek's disease virus, Newcastle disease virus, infectious laryngeal trachea. A recombinant herpesvirus encoding a virally derived antigenic polypeptide selected from the group consisting of a inflammatory virus, an infectious bronchitis virus, and a substantially homologous sequence.